Blebbistatin is a small molecule inhibitor discovered in a screen for inhibitors of nonmuscle myosin IIA. We have examined the specificity and potency of the drug by assaying its effects on the actin-activated MgATPase assay of diverse members of the myosin superfamily. Blebbistatin potently inhibits several striated muscle myosins as well as vertebrate nonmuscle myosin IIA and IIB with IC50 values ranging from 0.2 to 5 microM. Interestingly, smooth muscle, which is highly homologous to vertebrate nonmuscle myosin, is only poorly inhibited (IC50=80 microM). The drug potently inhibits Dictyostelium myosin II, but poorly inhibits Acanthamoeba myosin II. Blebbistatin did not inhibit representative myosin superfamily members from classes I, V, and X. In cells, blebbistatin has been shown to inhibit contraction of the cytokinetic ring. Blebbistatin has some photochemical properties that may affect its behavior in cells. We have examined the inhibition of nonmuscle myosins IIA and IIB proteins by several blebbistatin derivatives that have been synthesized. While the derivatives each inhibit the myosins with varying degrees of efficacy none of the derivatives gives more than a factor of five difference in the Ki for the two myosin isoforms in question. This means that they would be relatively ineffective at discriminating between the activities of these two myosin isoforms in in vivo studies.